The mycobacteria are a diverse collection of acid fast, gram-positive bacteria, some of which cause important human and animal diseases. In humans, the two most common mycobacteria-caused diseases are tuberculosis (TB) and leprosy, which result from infection with M. tuberculosis and M. leprae , respectively.
Tuberculosis displays all of the principal characteristics of a global epidemic disease. Currently, tuberculosis afflicts more than 35 million individuals worldwide and results in over 4 million deaths annually. In India, at any given time, almost 8 million people are reported to suffer from this disease and 500,000 deaths recorded. These figures may not cover the totality of those suffering from this disease in this country. Thus, tuberculosis appears to be a problem of major concern in India as also in many other countries of the world.
Tuberculosis is caused by M. tuberculosis, M. bovis, africanum and M. microti , the acid-fast, Gram positive, tubercle bacilli of the family Mycobacteriaceae. Some local pathogenic strains of M. tuberculosis have also been isolated from patients in Madras and other cities in India, which differ in some respects from M. tuberculosis H37Rv, which is a virulent strain.
In recent years, certain groups of individuals with AIDS have been found to have a markedly increased incidence of TB as well. It has now been shown that one group of mycobacteria which consists of M. avium, M. intracellulare and M. scrofulaceum, jointly known as MAIS complex, is responsible for disseminated disease in a large number of persons with AIDS (Kiehn et al., J. Clin. Microbiol., 21:168-173 (1985); Wong et al., Amer. J. Med., 78:35-40 (1985)).
Since Koch identified M. tuberculosis as the causative agent of tuberculosis in 1882, many scientific studies and public health efforts have been directed at diagnosis, treatment and control of this disease. However, characteristics of M. tuberculosis have hampered research to improve diagnosis and to develop more effective vaccines. In addition, the biochemical composition of the organism has made identification and purification of the cellular constituents difficult, and many of these materials once purified, lack sensitivity and specificity as diagnostic reagents. As a result, diagnostic and immunoprophylactic measures for mycobacterial diseases have changed little in the past half century. The conventional methods for the diagnosis of M. tuberculosis are troublesome and results are delayed.
Bacillus Calmette-Guerin (BCG), an avirulent strain of M. bovis (Calmette, A., Masson et Cie, Paris (1936)), is used extensively as a vaccine against tuberculosis. Though numerous studies have found that it has protective efficacy against tuberculosis (Luelmo, F., Am. Rev. Respir. Dis., 125, 70-72 (1982)) BCG has failed to protect against tuberculosis in several trials (WHO, Tech. Rep. Ser., 651:1-15 (1980)) for reasons that are not entirely clear (Fine, P., Tubercle, 65:137-153 (1984); Fine, et al., Lancet, (ii):499-502 (1986)).
The eradication with vaccination, early diagnosis, and efficient therapy is an important objective of the drive to combat mycobacterioses. The lacunae in the present knowledge of the biology of these pathogens--their make-up, their natural history, their physiology, biochemistry and immunological reactivities, highlights the need for attempts to unravel their weaknesses, so that more efficient ways to combat this disease can be devised. To develop more effective tools for the diagnosis and prevention of these diseases, it is important to understand the immune response to infection by mycobacterial pathogens. The mycobacterial components that are important in eliciting the cellular immune response are not yet well defined. The antibody and T-cell responses to infection or inoculation with killed mycobacteria have been studied in humans and in animals. Human patients with TB or leprosy produce serum antibodies directed against mycobacterial antigens. Although antibodies may have some function in the antimycobacterial immune response, the exact function remains to be clarified since no protective role can be ascribed to these antibodies. Protection against mycobacterial diseases involves cell-mediated immunity.
Mycobacteria do not produce any directly toxic substances and consequently their pathogenicity results from multiple factors involved in their interaction with the infected host. Intracellular parasitism probably depends on host cell trophic factors; it is conceivable that their short supply may be bacteriostatic and could play a role in the mechanism of mycobacterial dormancy.
It is generally understood that protective immunity in mycobacterial infection is mediated by specific T cells which activate macrophages into non-specific tuberculocidal activity. Evidence suggests that gamma-IFN triggers macrophages towards H.sub.2 O.sub.2 -mediated bacterial killing, but related or other macrophage activating factor (MAF) molecules may also be involved. The causes responsible for the inadequate bactericidal function at sites of abundant T cell proliferation have not yet been explained. Dissociation between delayed-type hypersensitivity (DTH) and protective immunity led to views that T-cells of a distinct subset or specificity could be responsible for the acquired resistance to mycobacterial infection. Alternatively, interference with protection may result from corollary cellular reactions, namely by suppressor T-cells and macrophages, or from the shifting of T-cells towards helper function for B-cells.
Unlike viral and some parasite pathogens which can evade host resistance by antigenic shift, mycobacteria have a resilient cell wall structure and can suppress host immune responses by the action of their immunodulatory cell wall constituents. Whilst the success of protective immunization towards other microbial pathogens mainly depends on quantitative parameters of immunity, it appears that mycobacterial immunomodulatory stimuli produce a regulatory dysfunction of the host immune system. This may not be possible to override simply by more resolute immunization using vaccines of complex composition such as whole mycobacteria (e.g. BCG). Perhaps mycobacteria did not evolve potent "adjuvant" structures to boost the host immunity but rather to subvert host defenses towards ineffective cellular reactions operating to the advantage of the pathogen. Vaccination with an attenuated pathogen such as BCG could amplify further immune responses but with limited protection of the host, the potential scope for immunization with defined antigens is yet to be explored.
The purification and characterization of individual antigenic proteins are essential in understanding the fundamental mechanism of the DTH reaction on the molecular level. The possible functional role of proteins of defined structure in the pathogenesis of mycobacterial diseases as well as for diagnostic purposes remains of great interest. Numerous groups have attempted to define mycobacterial antigens by standard biochemical and immunological techniques, and common as well as species specific antigens have been reported in mycobacteria (Minden, et al., Infect. Immun., 46:519-525 (1984); Closs, et al., Scand. J. Immunol., 12:249-263 (1980); Chaparas, et al., Am. Rev. Respir. Dis., 122:533 (1980); Daniel, et al., Microbiol. Rev., 42:84-113 (1978); Stanford, et al., Tubercle, 55:143-152 (1974); Kuwabara, S., J. Biol. Chem., 250:2556-2562 (1975)).
Very little information about the mycobacterial genome is available. Initially, basic studies were conducted to estimate the genome size, G+C content and the degree of DNA homology between the various mycobacterial genomes (Grosskinsky, et al., Infect. Immun., 57, 5:1535-1541 (1989); Garcia, et al., J. Gen. Microbiol., 132:2265-2269 (1986); Imaeda, T., Int. J. Sys. Bacteriol., 35, 2:147-150 (1985); Clark-Curtiss, et al., J. Bacteriol., 161 3:1093-1102 (1985); Baess, I. et al., B., Acta. Path. Microbial. Scand., (1978) 86:309-312; Bradley, S. G., Am. Rev. Respir. Dis., 106:122-124 (1972)). Recently, recombinant DNA techniques have been used for the cloning and expression of mycobacterial geners. Genomic DNA fragments of M. tuberculosis, M. leprae and some other mycobacterial species were used for the construction of lambda gtll phage (Young, et al., Proc. Natl. Acad. Sci., U.S.A., 82:2583-2587 (1985); Young, et al., Nature (London), 316:450-452 (1985)) or other vector-based recombinant gene libraries. These libraries were screened with murine monoclonal antibodies (Engers, et al., Infect. Immun., 48:603-605 (1985); Engers, et al., Infect. Immun., 51:718-720 (1986)) as well as polyclonal antisera and some immunodominant antigens were identified. The principal antigen among these being five 12, 14, 19, 65 & 71 kDa of M. tuberculosis (Young et al., Proc. Natl. Acad. Sci., U.S.A., 82:2583-2587 (1985); Shinnick et al., Infect. Immun., 55(7):1718-1721 (1987); Husson and Young, Proc. Natl. Sc. Acad., 84:1679-1683 (1987); and five 12, 18, 23, 36 & 65 kDa antigens of M. leprae (Young, et al., Nature (London), 316:450-452 (1985)). A few homologues of some of these antigens were also identified in some other mycobacterial species (e.g., BCG) (Yamaguchi et al., FEB 06511, 240:115-117 (1988); Yamaguchi et al., Infect. Immun., 57:283-288 (1989); Matsuo, et al., J. Bacteriol., 170, 9:3847-3854 (1988); Radford, et al., Infect. Immun., 56, 4:921-925 (1988); Lu, et al., Infect. Immun., 55, 10:2378-2382 (1987); Minden, et al., Infect. Immun., 53, 3:560-564 (1986); Horboe, et al., Infect. Immun., 52, 1:293-302 (1986); Thole, et al., Infect. Immun., 50, 3:800-806 (1985)). These antigens, however, are either intracellular or secreted molecules.
Although M. bovis BCG has been widely used as a vaccine against tuberculosis, the determination of the membrane-associated polypeptides of mycobacterium that are capable of inducing a protective immune response is highly desirable. The use of such a membrane-associated polypeptide or the DNA encoding it provides for the generation of recombinant vaccines, e.g., mycobacterial membrane-associated immunogens expressed in, for example, a virus or bacterium such as vaccinia virus, Salmonella, etc. used as a live carrier, or the display of non-mycobacterial immunogens on the surface of a cultivable mycobacterial strain which can be used as a live recombinant vaccine.
Accordingly, it is an object herein to provide methods for identifying and isolating nucleic acids encoding a membrane-associated polypeptide of mycobacteria.
Further, it is an object herein to provide membrane-associated polypeptides of mycobacteria and the nucleic acids encoding them.
Still further, it is an object herein to provide vaccines utilizing all or part of the membrane-associated polypeptide of a mycobacterium or the DNA encoding such membrane-associated polypeptide.
Still further, it is an object to provide reagents comprising said membrane-associated polypeptide with a mycobacterium or DNA encoding it useful in diagnostic assays for mycobacterial infection.
Still further, it is an object to provide a promoter sequence comprising the promoter of said membrane associated polypeptide, which can direct gene expression in mycobacteria as well as in other microorganisms such as E. coli.